Method for fabricating microparticles in emulsion by modification of the chemical composition of the dispersed phase after emulsification

ABSTRACT

The method for producing microparticles is characterized in that it comprises the following steps: preparation of an emulsion of a solution of a substance or a mixture of substances in a dispersing liquid wherein said substance or said mixture are substantially insoluble; incorporating to said dispersed phase a chemical agent substantially insoluble in the dispersing liquid so as to cause within the dispersed phase a chemical or physicochemical reaction responsible for the formation of microparticles which are then isolated.

The present invention relates essentially to a process for themanufacture of microparticles in emulsion by modification of thechemical composition of the disperse phase after emulsification.Advantageously, these microparticles are manufactured by a chemical orphysicochemical reaction within the disperse phase of an emulsion of asubstance or mixture of substances by modification of the chemicalcomposition of the disperse phase after emulsification, said reactionbeing caused by the incorporation of a chemical which is essentiallyinsoluble in the dispersing liquid.

The formation of microparticles by a chemical reaction is known forexample from the documents FR-A-2 444 497 Mars, FR-A-2 527 438 CNRS orelse 89 01221 BIOETICA, which describe the preparation of microcapsulesby the interfacial crosslinking of an aqueous solution of a protein or aprotein/polysaccharide mixture within a hydrophobic phase using adifunctional agent such as an acid dichloride.

It is also known to manufacture microparticles from solutions ofpolymers by physicochemical modifications, but these processes areso-called simple or complex coacervation processes (for example thedocument EP 0 273 823 A1 MERO ROUSSELOT SATIA), according to whether oneor two types of polymers are involved. In said document, thecoacervation reaction takes place in the continuous phase, resulting inthe formation of a membrane which deposits on the droplets of thedispersed hydrophobic liquid or on the particles of the dispersed solid.The process described in said document is furthermore limited to theencapsulation of a hydrophobic liquid or a solid, the encapsulation ofan aqueous phase being impossible.

The document U.S. Pat. No. 4,217,370 RAWLINGS describes a process forthe trapping of lipidic microdroplets dispersed in a continuous phasebased on a protein substance, which is precipitated by modification ofthe pH. This process thus consists in trapping droplets in a rigidifiedmatrix by precipitation of the continuous phase.

The document U.S. Pat. No. 4,187,194 WELLMAN describes a process forencapsulation by solvent evaporation.

The document U.S. Pat. No. 4,497,593 SIMKIN relates to a conventionalinterfacial crosslinking process in which no chemical modification iscarried out within the disperse phase.

The present invention makes it possible to manufacture microparticles bya different method from the conventional interfacial crosslinking orsimple and complex coacervation methods.

The object of the present invention is to manufacture microparticles, inparticular microcapsules, by an extremely simple and inexpensivemanufacturing process which has a good reproducibility and hence a goodreliability, which makes it possible to adjust the size of the particlesobtained within a wide range of values, and which, in particular,affords the possibility of avoiding the use of difunctional crosslinkingagents whose residual presence or whose reaction products are liable tojeopardize the biocompatibility of the microparticles or microcapsulesformed in this way, thereby making said process very competitive on theindustrial scale and preferentially suitable for use in cosmetics orpharmaceuticals or else foodstuffs. The object of the invention is tomodify the chemical or physicochemical composition of the disperse phasein order to manufacture microparticles, in particular microcapsules.

The present invention makes it possible to meet these demandssimultaneously.

Thus the present invention provides a process for the manufacture ofmicroparticles, in particular microcapsules, characterized in that anessentially homogeneous solution of a substance or mixture of substancesin a solvent is first prepared, an emulsion of the solution is producedin a dispersing liquid forming a continuous phase, in which saidsubstance or said mixture is essentially insoluble, and forming adisperse phase, and a chemical or physicochemical reaction is theninitiated in the disperse phase by modification of the in situ chemicalcomposition of said substance or said mixture in the disperse phasethrough the addition of an agent which is essentially insoluble in orimmiscible with the continuous phase, under the conditions of addition,thereby modifying the physicochemical state and resulting in theinsolubilization of the substance or mixture of substances and theindividualization of said microparticles, which are then recovered.

The subject of the present invention is based essentially on thepossibility, wholly novel to those skilled in the art, of being able tomodify the chemical composition of the disperse phase of an emulsionafter the dispersion has been produced, using agents which are insolublein or immiscible with the continuous phase.

In one advantageous variant, the agent which is essentially insoluble inor immiscible with the continuous phase, under the conditions ofaddition, and which it is desired to incorporate into the disperse phaseis present as a solution in a solvent which is miscible with the solventof the disperse phase and which has a lower affinity for the dispersingliquid of the continuous phase than for the disperse phase.

In one particular application, this operation makes it possible, totallyunexpectedly, to diffuse the reaction-initiating agent through ahydrophobic phase and into the droplets of a dispersed aqueous solution,whereas an aqueous solution of the same agent is completely ineffective.

As the substance or mixture of substances used to produce the solution,it is preferable to choose proteins, polysaccharides or nucleic acids,mixtures of proteins, mixtures of polysaccharides or mixtures ofproteins and polysaccharides, optionally with nucleic acids.

Proteins which may be mentioned are the following purified proteins:alpha-lactalbumin, beta-lactoglobulin, caseins, ovalbumin, animalalbumins, blood globulins, hemoglobin, fibrinogen, collagen,atelocollagen, gelatin, keratin, vegetable albumins, vegetableglobulins, glutenins and gliadin, and protein extracts derived frommilk, silk, cereals, leguminous plants, algae and fish.

Polysaccharides which may be mentioned are agar, agarose, agaropectin,carrageenans, alginates, pectins, amylose, amylopectin, starch, modifiedstarches, galactomannans (guar, carob), glucomannan, konjac, modifiedcelluloses, inulin, xanthan, dextran, curdlan, gellan, chitosan,chondroitin sulfates, hyaluronic acid, dermatan sulfate, heparansulfate, heparin and keratan sulfate.

Ribonucleic acid and deoxyribonucleic acid are envisaged as the nucleicacids.

The formation of covalent bonds, in particular esterifications andamidations, or the formation of ionic bonds between the ionizable groupsof a substance or mixture of substances is envisaged as the chemicalreaction.

A physicochemical reaction is understood essentially as meaningreactions such as coacervation, precipitation or desolvation reactions.

In another advantageous variant of the process, the latter ischaracterized in that the abovementioned agent which is essentiallyinsoluble in or immiscible with the continuous phase causes a phaseseparation within the initially homogeneous solution of the dispersephase, the gelling of said solution of the disperse phase or a loss ofsolubility of the disperse phase by condensation or polymerization.

In another advantageous variant of the process, the abovementioned agentwhich is essentially insoluble in or immiscible with the continuousphase, under the conditions of addition, is a non-solvent for thesubstance or mixture of substances solubilized in the solvent of thedisperse phase, or said agent causes a pH modification, or said agentcomprises at least one electrolyte, or said agent comprises at least onemolecule capable of reacting with the substance or substances dissolvedin the disperse phase of the emulsion.

In yet another advantageous variant, the abovementioned non-solvent forthe substance or mixture of substances solubilized in the solvent of thedisperse phase, which is essentially immiscible with the continuousphase under the conditions of addition, causes insolubilization of thedisperse phase, in particular gelling or coagulation.

In yet another particular variant, the abovementioned non-solvent isselected from an alcohol, in particular a C₁ -C₆ lower alcohol,preferably ethyl alcohol, or a ketone, in particular a C₂ -C₆ lowerketone, preferably acetone.

In a first advantageous embodiment, the following successive steps areperformed:

a) an aqueous solution of a substance selected from nucleic acids, aprotein or a polysaccharide, or various mixtures of these substances, isprepared;

b) a hydrophobic liquid is provided in which the abovementionedsubstance or substances are essentially insoluble;

c) the hydrophobic liquid and the aqueous phase are mixed to form anemulsion in which the aqueous solution is the disperse phase and thehydrophobic liquid is the continuous phase;

d) the non-solvent for the substance solubilized in the disperse phaseis added to the emulsion in proportions such that said non-solvent isessentially immiscible with the continuous phase, thereby formingmicroparticles by gelling or coagulation;

e) the microparticles formed by said gelling or said coagulation arecollected by physical separation means, for example by filtration,centrifugation or decantation; and

f) the cohesion of the microparticles is preferably strengthened bycrosslinking.

In a second advantageous embodiment, the first step is to produce anemulsion of an aqueous solution of a substance or mixture of substancescapable of undergoing, by a chemical or physicochemical reaction, avariation in physicochemical state represented by insolubilizationcaused by a pH modification, at a pH at which the physicochemical stateof said substance or said mixture of substances is that of a solutionwhose viscosity is appropriate for the production of an emulsion; the pHis then varied by the addition of the abovementioned insoluble orimmiscible agent, comprising a pH-modifying substance dissolved in anorganic solvent miscible with the aqueous phase, in order to initiatethe chemical or physicochemical reaction and bring the substance ormixture of substances into a physicochemical state which corresponds toinsolubilization of the substance or mixture and which results in theformation of physically individualized microparticles.

The pH-modifying substance used is generally an acid, a base or abuffer, depending on the desired pH value.

According to one advantageous characteristic of the processes accordingto the invention, the substance used if it is desired to render thedisperse phase of the emulsion alkaline is a solution of sodiumhydroxide or potassium hydroxide in an alcohol such as methanol orethanol, used pure or containing 5 to 10% (w/v) of water, or else apolyol such as glycerol or a polyethylene glycol. According to apreferred characteristic, the solution contains between 0.5 and 10% ofsodium hydroxide in 95% (w/v) ethanol.

According to another advantageous characteristic of the processesaccording to the invention, the substance used if it is desired toacidify the disperse phase of the emulsion is a solution of amonocarboxylic or polycarboxylic acid which may or may not carry alcoholgroups, such as acetic acid, citric acid, lactic acid, tartaric acid,succinic acid or malic acid, or a mineral acid such as hydrochloricacid, in an alcohol such as methanol or ethanol, used pure or containing5 to 10% of water, or else in a polyol such as glycerol or apolyethylene glycol. According to a preferred characteristic, the acidsolution consists of 95% ethanol containing between 1 and 10% (v/v) ofacetic acid.

According to another feature, the reaction caused in situ by the changeof pH in the droplets of the disperse phase is a physicochemicalcoacervation reaction.

In this case, the process for the manufacture of the microparticles isadvantageously as follows:

a) an aqueous solution of a substance selected from nucleic acids, apolysaccharide or a protein, or various mixtures of these substances, isprepared, the pH being chosen so that the substance or mixture ofsubstances forms an essentially homogeneous solution whose viscosity iscompatible with the production of an emulsion;

b) a hydrophobic liquid is provided in which the abovementionedsubstance or substances are essentially insoluble;

c) the hydrophobic liquid and the aqueous phase are mixed to form anemulsion;

d) either a solution of an alkaline substance in an organic liquidmiscible with the aqueous phase is added to the emulsion, if it isdesired to render the aqueous phase alkaline, or a solution of an acidsubstance in an organic liquid miscible with the aqueous phase is addedto the emulsion, if it is desired to acidify the aqueous phase;

e) after a predetermined period of time required to effect thecoacervation of the initially solubilized substance or substances, themicroparticles formed are collected by centrifugation or filtration; and

f) the cohesion of the microparticles is preferably strengthened bycrosslinking, crosslinking being preferred in the majority of cases.

A number of reagents may be mentioned as examples ofagents--conventional to those skilled in the art--for crosslinkingpolyamino, polycarboxylic or polyhydroxylic compounds or compoundshaving a mixture of these types of groups; said reagents can beseparated into two types.

The first type consists of difunctional agents which form a bridgebetween the reactive groups of the substance or substances constitutingthe microparticles, and remain in the reaction product. Formaldehyde,glutaraldehyde and dialdehydes in general, acid dichlorides, aciddianhydrides, diisocyanates, diimidoesters, bischloroformates andsuccinimides belong to reagents of this type.

The second type of reagent is made up of agents which do not form abridge but which activate the carboxyl groups, in particular, so thatthey can form amide bonds with the amino groups or ester bonds with thealcohol groups of the substance or mixture of substances used tomanufacture the microparticles. The azide methods such as described inthe documents EP-A-0301977 BIOETICA and WO 90/12055 BIOETICA, or themethods using carbodiimides, for example, are representative of thistype of method.

In the case where it is desired to prepare microparticles of perfectbiocompatibility, which is absolutely essential for use inpharmaceuticals, it will be particularly preferable to use methods ofthe second type, which, by permitting the formation of amide or esterbonds directly between on the one hand the carboxyl groups and on theother hand the amino or alcohol groups of the substance or substancesconstituting the microparticles, make it possible quite remarkably toobtain microparticles consisting solely of the substance or substancesused to produce the emulsion, to the exclusion of any crosslinkingagent.

In one particular variant, the protein used is collagen; in fact, thisprotein is essentially soluble at a pH below 4 and precipitates at pHvalues above 4. A collagen solution is therefore manufactured at a pH ofabout 3.5. An emulsion is produced by dispersion of the collagensolution in a hydrophobic liquid. The droplets of the collagen solutionare rendered alkaline by the addition of an alkaline alcoholic solutionto the emulsion, causing precipitation of the collagen and henceindividualization of collagen microparticles, which can be recovered bycentrifugation or filtration and which can subsequently be subjected tocrosslinking by the abovementioned methods.

According to one advantageous characteristic of the invention, theconcentration of the collagen solution is between 0.1 and 2%, preferablyabout 0.5% (w/v).

In a second particular variant, the protein used is a collagenderivative, preferably atelocollagen.

According to one advantageous characteristic of the invention, theconcentration of the atelocollagen solution is between 0.3 and 4% (w/v).

In a third particular variant, the polysaccharide used is aglycosaminoglycan well known to those skilled in the art.Glycosaminoglycans are described in particular in the Applicant'sdocument EP-A-0318154 of the prior art. The polysaccharide used can alsobe chitosan. Chitosan is a polyionic polymer which is only soluble inthe aqueous phase at pH values below 5. When the pH of a chitosansolution is brought to a value close to neutrality, the polymerprecipitates to form perfectly water-insoluble fibrils. This propertycan be utilized for the production of microcapsules according to theinvention. A chitosan solution is prepared at acid pH, an emulsion isproduced in a hydrophobic liquid and the pH of the disperse phase ismodified by the addition of an alcoholic solution of a base to theemulsion. Precipitation in situ permits the formation of readilyrecoverable microparticles, which can subsequently be chemicallycrosslinked if necessary.

According to one advantageous characteristic of the invention, it ispreferable to use a highly deacetylated chitosan whose molecular weightis very high, preferably greater than 300,000 daltons. "Highlydeacetylated" is preferably understood as meaning a residual degree ofacetylation of less than about 5%, preferably 3.5%. Such highlydeacetylated chitosans are commercially available.

According to one advantageous characteristic of the invention, theconcentration of the chitosan solution is between 0.2 and 10%,preferably about 1% (w/v).

According to a second feature of the formation of spheres by varying thepH of the disperse phase of an emulsion, microcapsules are formed, oncethe emulsion has been produced, by the initiation of a chemical reactionbetween the reactive groups of a substance or mixture of substances.

The chemical reaction can be for example a transacylation reaction whichtakes place at alkaline pH between a polysaccharide carrying esterifiedcarboxyl groups and either a polyamino substance, for example a protein,or a polyhydroxylic substance, for example a polysaccharide.

In this case, the process for the manufacture of the microparticles isadvantageously as follows:

a) a neutral, i.e. unreactive, aqueous solution is prepared whichcontains on the one hand a polysaccharide carrying esterified carboxylgroups and on the other hand either a polyamino substance, for example aprotein, or a polyhydroxylic substance, for example a polysaccharide;

b) a hydrophobic liquid is provided in which the esterifiedpolysaccharide and the polyamino or polyhydroxylic substance areessentially insoluble;

c) the hydrophobic liquid and the aqueous solution are mixed to form anemulsion;

d) a solution of an alkaline substance in an organic liquid misciblewith the aqueous phase is added to the emulsion, making it possible toobtain physicochemical conditions favorable to the reaction between thepolyamino substance or the polyhydroxylic substance and thepolysaccharide carrying esterified carboxyl groups; and

e) after a predetermined period of time required to effect atransacylation reaction, thereby forming microparticles, in particularmicrocapsules, the emulsion is neutralized, preferably by the addition,to the emulsion, of a solution of an acid substance in an organic liquidmiscible with the aqueous phase, which neutralizes and stabilizes themicroparticles formed, in particular the microcapsules formed.

In one variant, the emulsion formed is an emulsion of the aqueoussolution as the disperse phase in the hydrophobic liquid as thecontinuous phase.

In a second mode of utilizing the invention, the modification ofchemical composition induced in situ after production of the emulsion isa modification of the concentration of certain electrolytes.

A number of polysaccharides associate specifically with ions or polyionsto form gels.

In particular, a number of polyanionic polysaccharides associatespecifically with cations or polycations, more particularly withmonovalent cations such as potassium and sodium, or divalent cationssuch as calcium, magnesium, strontium or barium. This associationresults in the formation of gels of greater or lesser susceptibility tobreaking, according to the nature of the polysaccharide and the cationand to the concentrations used.

In one particular variant, a mixture of polysaccharides will be producedso that the cohesion of the gel formed can be modulated.

Kappa- and iota-carrageenans, pectins with a low degree of methylation,alginates and gellan gum are chosen as the polysaccharides which arepreferably used.

In one variant, the polysaccharide is gellan gum, which is a polyanionicpolysaccharide of very high molecular weight (at least 500,000 daltons),manufactured by microorganisms. When solubilized in water, gellan givesa low-viscosity solution with which it is possible to produce anemulsion by dispersion in a hydrophobic phase. When such an emulsion isheated to a temperature above 75° C. and cations are added by a processaccording to the invention, the droplets of the disperse phase undergogelling. After returning to room temperature, the spheres formed in thisway can be recovered and then chemically crosslinked if necessary.

According to one advantageous characteristic of the process according tothe invention, the concentration of the gellan solution is between 0.1and 5% and preferably about 0.6% (w/v).

According to another characteristic of the manufacturing process, anethanolic solution is produced which contains calcium, magnesium, sodiumor potassium ions so that the solution is brought to an ionconcentration of between 1 and 400 mM, preferably of between 5 and 10 mMfor calcium and magnesium ions and preferably of between 150 and 250 mMfor sodium and potassium ions.

In another variant, the polysaccharide used is a kappa-carrageenan or aniota-carrageenan, which gels in the presence of potassium or calciumions respectively.

According to one advantageous characteristic of the process according tothe invention, the carrageenan is dissolved in the cold, at neutral oralkaline pH, in concentrations of between 0.1 and 5% (w/v), preferablyabout 1.5%. The temperature is raised to around 80° C. and an emulsionis then produced by dispersion in a hydrophobic liquid. Gelling of thedroplets is then caused by the addition of an alcoholic solutioncontaining potassium or calcium ions, according to whether the aqueoussolution contains kappa- or iota-carrageenan.

In a third variant, the polysaccharide used is a pectin with a lowdegree of methylation, which forms solutions gellable in the presence ofcalcium ions.

According to one advantageous characteristic, a pectin solution with aconcentration of between 0.1 and 10%, preferably of the order of 1 to 2%(w/v), is produced in the cold at neutral pH. An emulsion is produced bydispersion in a hydrophobic liquid. According to the invention, calciumions are then added in the form of an alcoholic solution of calciumchloride.

In a last variant, the same type of process is carried out starting froma solution containing from 1 to 5% (w/v) of sodium alginate.

Finally, according to yet another feature, the present invention furtherrelates to a composition such as a cosmetic composition or apharmaceutical composition, or a food composition, characterized in thatit comprises microparticles, in particular microcapsules, obtained bythe abovementioned process.

Other objects, characteristics and advantages of the invention willbecome clearly apparent from the following explanatory descriptionreferring to several Examples of the invention, which are given simplyby way of illustration and cannot therefore in any way limit the scopeof the invention. The percentages are given by weight in the Examples,unless indicated otherwise.

EXAMPLE 1 OF THE INVENTION

Manufacture of microparticles of atelocollagen

a) Preparation of the aqueous phase

A 2% (w/v) solution of bovine atelocollagen is prepared, the pH of whichis adjusted to 4.

b) Emulsification

40 ml of this aqueous phase are emulsified, by mechanical agitation fora few minutes, in 250 ml of 2-ethylhexyl cocoate containing 2% v/v ofSpan 85® as the continuous phase.

c) Alkalization

Twice 10 ml of 2% (w/v) sodium hydroxide in 95% ethanol are added 10 minapart. The reaction is allowed to proceed for 10 min after eachaddition.

d) Washes

The microparticles in the form of spheres are collected bycentrifugation and then washed in several baths of 95% ethanol.

e) Crosslinking

The spheres are crosslinked by incubation for 24 h at 4° C. in a bath ofdimethylformamide containing 0.5% (v/v) of diphenylphosphoryl azide, andthen for 4 h at room temperature in borate buffer of pH 8.9 (sodiumtetraborate 0.04M, boric acid 0.04M).

After washes with water, the microparticles thus obtained can belyophilized.

EXAMPLE 2 OF THE INVENTION

Manufacture of microparticles of chitosan

a) Preparation of the aqueous phase

A 1% (w/v) solution of highly deacetylated HMW chitosan (AbetTechnologies) in 1.25% acetic acid, pH 4, is prepared.

b) Emulsification

An emulsion is produced by dispersion of 50 ml of this solution in 250ml of isopropyl myristate containing 2% (v/v) of Span 85®. The emulsionis agitated mechanically for 10 min.

c) Alkalization

16 ml of 2% w/v sodium hydroxide in 95% ethanol are added and agitationis continued for 15 min.

d) Washes

The microparticles are recovered by centrifugation and washed in severalbaths of ethanol and then water.

e) Crosslinking

The microparticles can be crosslinked as described in Example 1.

EXAMPLE 3 ACCORDING TO THE INVENTION

Manufacture of microparticles of mean diameter 150 μm from human serumalbumin (HSA) and propylene glycol alginate (PGA)

a) preparation of the aqueous phase

A solution in distilled water containing 20% of HSA (Centre deTransfusion Sanguine, Strasbourg) and 1% of a PGA having a degree ofesterification of between 80 and 85% (Kelcoloid S®, KELCO International)is prepared by magnetic agitation for 10 min at room temperature.

b) Emulsification

6 ml of this aqueous phase, used as the disperse phase, are emulsified,by mechanical agitation for 5 min at 2000 rpm, in 40 ml of isopropylmyristate containing 2% v/v of Span 85® as the continuous phase.

c) Alkalization

2 ml of a 2% w/v solution of sodium hydroxide in 95% ethanol are addedto the emulsion, with agitation, and the transacylation reaction isallowed to proceed for 15 min to produce microparticles.

d) Acidification

2 ml of a 7.6% v/v solution of acetic acid in 95% ethanol are added tothe reaction medium, with agitation. Agitation is maintained for afurther 15 min to permit the neutralization of the microparticlesformed.

e) Washes

The microparticles are separated off by centrifugation and subsequentlywashed by resuspension in 95% ethanol containing 2% of Tween 20®, thenin 95% ethanol and then in distilled water.

The microparticles can then be frozen and lyophilized.

This gives transparent spheres with a mean size of 150 μm. Afterlyophilization, rehydration of the powder obtained shows that themicroparticles are intact and resume their spherical shape.

Stability tests in various media containing or not containing proteases

In test tubes, 25 mg samples of lyophilized microparticles arerehydrated by the addition of 1 ml of distilled water, after which 7.5ml of the following different media are added:

distilled water

a solution of acid pH (1.2) to which pepsin (artificial gastric medium,USP XXI) may or may not have been added

a solution of slightly alkaline pH (7.5) to which trypsin (0.25% w/v)may or may not have been added.

The tubes are incubated at 37° C. The stability of the microparticles isstudied by microscopic examination. The lysis time is the time afterwhich all the microparticles have disappeared from the medium.

Results

The microparticles prepared according to this Example are stable formore than 3 d in distilled water and in the solutions of pH 1.2 or pH7.5. They are degraded by proteases: in 15 min by pepsin and in 25 minby trypsin.

EXAMPLE 4 OF THE INVENTION

Manufacture of microparticles from gelatin and PGA

Preparation of the aqueous phase

8 ml of an aqueous solution of type B gelatin, bloom 150, at aconcentration of 10% and PGA at a concentration of 1% are prepared at atemperature of 40° C.

Emulsification

In a thermostated vessel at 40° C., 6 ml of this aqueous phase areemulsified in 40 ml of isopropyl myristate containing 2% of Span 85 andpreheated to a temperature of 40° C. (agitation speed: 2000 rpm).

Alkalization

Neutralization and washes are then effected as described in Example 3.The microparticles appear as spheres of mean diameter 1 mm. Afterlyophilization, they give a white powder which is readily rehydratable.

EXAMPLE 5 OF THE INVENTION

Manufacture of microparticles from atelocollagen, chondroitin sulfateand PGA

Preparation of the aqueous phase

PGA is added at a concentration of 0.7% to a solution containing 1.6% ofatelocollagen and 0.6% of chondroitin sulfate in a phosphate buffer ofpH 7.4. The protocol described in Example 3 is then applied to thisaqueous solution.

Microparticles of mean diameter 600 μm are obtained.

EXAMPLE 6 OF THE INVENTION

Manufacture of microparticles from a whey protein concentrate and PGA

Preparation of the aqueous phase

160 mg of PGA and 3.2 g of whey protein concentrate (Prosobel S65E®, BelIndustries) are dissolved in 16 ml of distilled water.

The protocol described in Example 3 is then repeated using 12 ml of theabove solution as the aqueous phase and doubling all the volumes of thevarious reagents.

This gives spherical microparticles of mean diameter 500 μm and ofgranular content, which are intact after lyophilization.

EXAMPLE 7 OF THE INVENTION

Manufacture of microparticles from ovalbumin and pectin

Preparation of the aqueous phase

240 mg of apple pectin (FLUKA, esterification: 70 to 75%) and 800 mg ofovalbumin are dissolved in 8 ml of distilled water.

The protocol described in Example 3 is then applied using 6 ml of theabove solution, replacing the isopropyl myristate with fluid paraffinoil and doubling the volumes of the alkaline and acid solutions.

This gives microparticles of mean size 200 μm and of granular content.

EXAMPLE 8 OF THE INVENTION

Manufacture of microparticles from carboxymethyl cellulose (CMC) and PGA

Preparation of the aqueous phase: 80 mg of CMC (CMC 7 LF, degree ofsubstitution: 0.7, HERCULES) and 320 mg of PGA are dissolved in 8 ml ofdistilled water by magnetic agitation for 15 min at 40° C.

The protocol described in Example 3 is then applied using 6 ml of theabove solution.

This gives granular microparticles of mean size 1.8 mm.

EXAMPLE 9 OF THE INVENTION

Manufacture of microparticles of atelocollagen

a) Preparation of the aqueous phase

A 2% (w/v) solution of bovine atelocollagen is prepared, the pH of whichis adjusted to 3.7 in the manner well known to those skilled in the art,especially from the Applicant's previous documents such as EP-A-0318154,to which those skilled in the art may refer.

b) Emulsification

50 ml of this phase are emulsified, by mechanical agitation for a fewminutes, in 200 ml of 2-ethylhexyl cocoate as the continuous phase.

c) Addition of the non-solvent

400 ml of 95° ethyl alcohol are added with continued agitation. Theagitation is stopped after a few seconds, causing microparticles toprecipitate.

d) Washing

The microparticles obtained are collected by filtration and then washedin several baths of 95° ethanol.

e) Crosslinking

The microparticles are preferably crosslinked by incubation for 24 h at4° C. in a bath of dimethylformamide containing 0.5% (v/v) ofdiphenylphosphoryl azide and then for 4 h at room temperature in boratebuffer of pH 8.9.

After washing with water, the resulting microparticles can belyophilized.

EXAMPLE 10 OF THE INVENTION

Manufacture of microparticles of atelocollagen

In this Example, the procedure described in Example 9 is followed exceptthat the ethyl alcohol is replaced with acetone as the non-solvent forprecipitating the microparticles.

After the microparticles have been washed several times in several bathsof acetone, the crosslinking can be carried out as described in Example9.

After washing with water, the microparticles can also be lyophilized.

The invention covers any means which constitute technical equivalents ofthe means described, as well as the various combinations thereof. Theinvention also covers any characteristic which appears novel comparedwith any state of the art, on the basis of the description taken as awhole, i.e. including the claims.

What is claimed is:
 1. A process for the manufacture of microparticlescomprising:a) preparing an essentially homogenous solution of asubstance or mixture of substances in a first solvent, b) producing anemulsion of this homogenous solution in a dispersing liquid forming acontinuous phase, in which said substance or said mixture of substancesis essentially insoluble, and forming a disperse phase, c) initiating achemical or physicochemical reaction in the disperse phase bymodification of the in situ chemical composition of said substance orsaid mixture or substances in the disperse phase by adding thereto anagent which is essentially insoluble in or immiscible with thecontinuous phase, under the conditions of addition, thereby modifyingthe physicochemical state of said disperse phase resulting in theinsolubilization of the substance or mixture of substances in thedisperse phase and the individualization of said microparticles, in thepresence of the first solvent.
 2. The process of claim 1, wherein theagent which is essentially insoluble in or immiscible with thecontinuous phase, under the conditions of addition, which is added tothe disperse phase is added as a solution in a second solvent which ismiscible with the first solvent of the disperse phase and which has alower affinity for the dispersing liquid of the continuous phase thanfor the disperse phase.
 3. The process of claim 1, wherein saidsubstance or mixture of substances is selected from the group consistingof nucleic acids, a protein, a polysaccharide, and any mixture thereof.4. The process of claim 1, wherein a chemical reaction is initiatedcomprising the formation of covalent bonds.
 5. The process of claim 1,wherein said chemical reaction providing the formation of covalent bondsis selected from the group consisting of an esterification and anamidation.
 6. The process of claim 1, wherein said chemical reactioncomprises the formation of ionic bonds between the ionizable groups ofsaid substance comprising ionizable groups.
 7. The process of claim 6,wherein said chemical reaction comprising ionizable groups is selectedfrom the group consisting of nucleic acids, a protein, a polysaccharideand any mixture thereof.
 8. The process of claim 1, wherein aphysicochemical reaction is initiated selected from the group consistingof a coacervation reaction, and a precipitation reaction.
 9. The processof claim 1, wherein said agent which is essentially insoluble in orimmiscible with the continuous phase causes a physicochemical reactionselected from the group consisting of a phase separation within theinitially homogenous solution of the disperse phase, the gelling of saidsolution of the disperse phase and a loss of solubility of the dispersephase by condensation or polymerization of said substance.
 10. Theprocess of claim 1, wherein said agent which is essentially insoluble inor immiscible with the continuous phase, under the conditions ofaddition, is a non-solvent for the substance or mixture of substancessolubilized in the first solvent of the disperse phase.
 11. The processof claim 1, wherein said agent which is essentially insoluble in orimmiscible with the continuous phase, under the conditions of addition,causes a pH modification of the disperse phase.
 12. The process of claim1, wherein said agent which is essentially insoluble in or immisciblewith the continuous phase, under the conditions of addition, comprisesat least one electrolyte.
 13. The process of claim 1, wherein said agentwhich is essentially insoluble in or immiscible with the continuousphase, under the conditions of addition, comprises at least one moleculecapable of reacting with the substance or substances dissolved in thedisperse phase of the emulsion.
 14. The process of claim 10, wherein thenon-solvent for the substance or mixture of substances solubilized inthe solvent of the disperse phase causes insolubilization of thesubstance or mixture of substances of the disperse phase.
 15. Theprocess of claim 14, wherein said insolubilization is selected from thegroup consisting of a gelling and a coagulation.
 16. The process ofclaim 14, wherein said non-solvent is selected from the group consistingof an alcohol and a ketone.
 17. The process of claim 16, wherein saidalcohol is a C₁ -C₆ lower alcohol and said ketone is a C₂ -C₆ lowerketone.
 18. The process of claim 17, wherein said alcohol is ethanol andsaid ketone is acetone.
 19. The process of claim 1, which is a processfor the manufacture of microcapsules.
 20. A process for the manufactureof microparticles, comprising:a) preparing an aqueous solution of asubstance selected from the group consisting of nucleic acids, aprotein, a polysaccharide and any mixture thereof; b) providing ahydrophobic liquid in which said substance or substances are essentiallyinsoluble; c) mixing the hydrophobic liquid and the aqueous solution toform an emulsion in which the aqueous solution is a disperse phase andthe hydrophobic liquid is a continuous phase; d) adding to the emulsiona non-solvent for the substance solubilized in the disperse phase indetermined proportions for having said non-solvent essentiallyimmiscible with the continuous phase and for forming microparticles fromthe disperse phase by insolubilization of said substance or substancesin the disperse phase, and e) recovering said microparticles by physicalseparation.
 21. The process of claim 20, wherein said insolubilizationis selected from the group consisting of a gelling and a coagulation.22. The process of claim 20, wherein said physical separation isselected from the group consisting of a filtration, a centrifugation anda decantation.
 23. The process of claim 20, further comprisingcrosslinking the microparticles with a crosslinking agent.
 24. Theprocess of claim 20, wherein said insolubilization comprises adding saidinsoluble or immiscible agent comprising a pH-modifying substancedissolved in an organic solvent miscible with the disperse phase. 25.The process of claim 24, wherein the pH-modifying substance is selectedfrom the group consisting of an acid, a base and a buffer, depending onthe required pH value.
 26. The process of claim 20, for the preparationof microcapsules.
 27. A process for the manufacture of microparticles,comprising:a) preparing an aqueous solution of a substance selected fromthe group consisting of nucleic acids, a protein, a polysaccharide andany mixture thereof, said aqueous solution having a pH adapted forforming an essentially homogeneous solution of said substance or mixtureof substances with a viscosity compatible with the production of anemulsion; b) providing a hydrophobic liquid in which the substance ormixture of substances are essentially insoluble; c) mixing thehydrophobic liquid and the aqueous solution to form an emulsion in whichthe aqueous solution is a disperse phase and the hydrophobic liquid is acontinuous phase; d) adding to the emulsion a solution of a substanceselected from the group consisting of an alkaline substance and an acidsubstance, in an organic liquid miscible wit the aqueous phase, therebyinsolubilizing said substance or substances resulting in formation ofmicroparticles from said disperse phase; and e) recovering saidmicroparticles.
 28. The process of claim 27, further comprisingcrosslinking said microparticles with a crosslinking agent.
 29. Theprocess of claim 28, wherein said crosslinking agent is a bifunctionalcrosslinking agent capable of reacting with said substance orsubstances, comprising carboxylic groups, amine groups and hydroxylgroups, and an agent capable of activating the carboxylic groups of saidsubstance or substances to form covalent bonds selected from the groupconsisting of amide bonds with amine groups, and ester bonds withhydroxyl groups of said substance or substances.
 30. The process ofclaim 29, wherein said bifunctional crosslinking agent is selected fromthe group consisting of formaldehyde, glutaraldehyde, di-aldehydes, aciddichlorides, di-acids anhydrides, di-isocyanates, di-imidoesters,bis-chloroformates and succinimides, and said agent activating carboxylgroups is selected from the group consisting of a carbodiimide and anazide.
 31. The process of claim 1, wherein the substance is a proteinselected from the group consisting of collagen and atelocollagen, andsaid polysaccharide is selected from the group consisting ofglycosaminoglycan and chitosan.
 32. The process of claim 20, wherein theprotein is selected from the group consisting of collagen,atelocollagen, said polysaccharide is selected from the group consistingof gllycosaminoglycan and chitosan.
 33. The process of claim 27, whereinthe protein is selected from the group consisting of collagen,atelocollagen, said polysaccharide is selected from the group consistingof glycosaminoglycan and chitosan.
 34. The process of claim 27comprising:a) preparing a neutral, unreactive aqueous solutioncontaining a polysaccharide carrying esterified carboxyl groups and asubstance selected from the group consisting of a polyamino substance,and a polyhydroxylic substance; b) providing a hydrophobic liquid inwhich the esterified polysaccharide and said substance are essentiallyinsoluble; c) mixing the hydrophobic liquid and the aqueous solution toform an emulsion constituting a disperse phase; d) adding to theemulsion a solution of an alkaline substance in an organic liquidmiscible with the aqueous phase thereby initiating a transacylationreaction in said disperse phase between the polysaccharide carryingesterified carboxyl groups and said substance; and e) neutralizing saidemulsion by adding to the emulsion of a solution of an acid substance inan organic liquid miscible with the aqueous phase, thereby stabilizingmicroparticles formed thereby.
 35. The process of claim 34, comprisingmanufacturing microcapsules.
 36. The process of claim 35, wherein saidsubstance is a polysaccharide which is capable of associatingspecifically with an ion entity to form a gel.
 37. The process of claim36, wherein said polysaccharide is selected from the group consisting ofkappa-carrageenan, iota-carrageenan, a pectin with a low degree ofmethylation, an alginate and a gellan gum having a high molecular withof a least 500,000 daltons.
 38. The process of claim 37, wherein gellanis used in an aqueous solution at a concentration of between 0.1 and 5%weight/volume.
 39. The process of claim 38, wherein gellan is used in aaqueous solution at a concentration of about 0.6% weight/volume.
 40. Theprocess of claim 39, wherein said ionic entity is present as anethanolic solution having an ion concentration ranging between 1 and 400mM.
 41. The process of claim 40, wherein said ionic entity is selectedfrom the group consisting of calcium, magnesium, sodium and potassium.42. The process of claim 36, wherein said polysaccharide is selectedfrom the group consisting of kappa-carrageenan and iota-carrageenanwhich is gellable by an ionic entity selected from the group consistingof potassium ions and calcium ions; said process comprising solving thecarrageenan in the cold in an aqueous solution initially brought at a pHselected from the group consisting of a neutral pH and an alkaline pH,at a concentration ranging between 0.1 and 5% in weight/volume, raisingthe temperature to about 80° C., producing an emulsion by dispersion ofsaid carrageenan aqueous solution in a hydrophobic liquid comprisingdroplets of said carrageenan aqueous solution dispersed in saidhydrophobic liquid; and gelling said droplets by adding to the emulsionan alcoholic solution containing said ionic entity, therebymanufacturing said microparticles by gelling said carrageenan.
 43. Theprocess of claim 36, wherein said polysaccharide is a pectin with a lowdegree of methylation which is gellable in the presence of calcium ions,said process comprising preparing a pectin solution at a concentrationranging between 0.1 and 10% weight/volume in the cold at a substantiallyneutral pH, producing an emulsion of said pectin solution by dispersionin a hydrophobic liquid to provide droplets of said pectin solution insaid hydrophobic liquid, and gelling said droplets by adding analcoholic solution containing calcium ions, thereby manufacturing pectinbased microparticles.
 44. The process of claim 36, wherein thepolysaccharide is a 1 to 5% weight/volume solution of sodium alginate.45. Microparticles made from a substance or mixture of substances whichhas been first solubilized in a first solvent dispersed in a dispersionliquid to form an emulsion before being insolubilized to form saidmicroparticles.
 46. Microparticles according to claim 45, made from saidsubstance or mixture of substances comprising carboxylic groups, and atleast one of amine groups and alcohol groups, which are crosslinked dueto activation of the carboxylic groups by an activating agent which doesnot form a bridge, thereby forming an amide bond between a carboxylicgroup and an amino group or an ester bond between a carboxylic group andan alcohol group.
 47. Microparticles according to claim 46 whichcomprise a reaction product between a polysaccharide bearing esterifiedcarboxylic groups and at least one said substance selected from thegroup consisting of a polyamine substance and a polyhydroxy substance.48. Microparticles according to claim 45, wherein said microparticleshave an inner filled space thereby constituting microcapsules and have awall essentially constituted by a reaction product between apolysaccharide bearing esterified carboxylic groups and at least onesaid substance selected from the group consisting of a polyaminesubstance and a polyhydroxy substance.
 49. Microparticles according toclaim 45, wherein said substance or mixture of substances is selectedfrom the group consisting of a protein, a polysaccharide, a nucleicacid, a mixture of proteins, a mixture of polysaccharides, a mixture ofproteins and polysaccharides, a mixture of a protein with a nucleicacid, a mixture of polysaccharide with a nucleic acid and a mixture ofprotein, polysaccharide and nucleic acid.
 50. Microparticles accordingto claim 45, wherein said substance of mixture of substances is aprotein selected from the group consisting of alpha-lactalbumin,beta-lactoglobulin, a casein, ovalbumin, an animal albumin, a bloodglobulin, hemoglobin, fibrinogen, collagen, atelocollagen, gelatin,keratin, a vegetable albumin, a vegetable globulin, glutenin, gliadin, aprotein extract derived from milk, silk, a protein extract derived froma cereal, a protein extract derived from a leguminous plant, a proteinextract from an algae and a protein extract derived from fish. 51.Microparticles according to claim 45, wherein said substance or mixtureof substances is a polysaccharide selected from the group consisting ofagar, agarose, agaropectin, a carrageenan, an alginate, a pectin,amylose, amylopectin, starch, a modified starch, a galactomannan, aglucomannan, konjac, a modified cellulose, inulin, xanthan, dextran,curdlan, gellan, chitosan, a chondroitin sulfate, hyaluronic acid,dermatan sulfate, heparan sulfate, heparin and keratan sulfate. 52.Microparticles according to claim 45, wherein said substance or mixtureof substances is selected from the group consisting of a gellablepolysaccharide and a coagulatable polysaccharide which is gelled orcoagulated.
 53. Microparticles according to claim 45, wherein saidsubstance or mixture of substances is a polysaccharide selected from thegroup consisting of carrageenan kappa, carrageenan iota, a pectin with alow degree of methylation, an alginate and gellan gum. 54.Microparticles according to claim 45, wherein said substance or mixtureof substances is a propylen glycol alginate.
 55. Microparticlesaccording to claim 45, wherein said microparticles are lyophilized. 56.A composition selected from the group consisting of a cosmeticcomposition, a pharmaceutical composition and a food composition, saidcomposition comprising microparticles as defined in claim
 45. 57. Aproduct produced by the process of claim
 1. 58. A product produced bythe process of claim
 20. 59. A product produced by the process of claim27.
 60. A composition selected from the group consisting of a cosmeticcomposition, a pharmaceutical composition and a food compositioncomprising microparticles as obtained by the process of claim
 1. 61. Acomposition selected from the group consisting of a cosmeticcomposition, a pharmaceutical composition and a food compositioncomprising microparticles as prepared by the process of claim
 20. 62. Acomposition selected from the group consisting of a cosmeticcomposition, a pharmaceutical composition and a food compositioncomprising microparticles as prepared by the process of claim
 27. 63.The process of claim 23, wherein said crosslinking agent is abifunctional crosslinking agent capable of reacting with said substanceor substances, comprising carboxylic groups, amine groups and hydroxylgroups, and an agent capable of activating the carboxylic groups of saidsubstance or substances to form covalent bonds selected from the groupconsisting of amide bonds with amine groups, and ester bonds withhydroxyl groups of said substance or substances.
 64. The process ofclaim 63, wherein said bifunctional crosslinking agent is selected fromthe group consisting of formaldehyde, glutaraldehyde, di-aldehydes, aciddichlorides, di-acid anhydrides, di-isocyanates, di-imidoesters,bis-chloroformates and succinimides, and said agent activating carboxylgroups is selected from the group consisting of a carbodiimide and anazide.
 65. A process according to claim 27, further comprisinglyophilizing the microparticles.
 66. The process of claim 1, wherein thehomogeneous solution comprises a mixture of human serum albumin andpropylene glycol alginate.
 67. The process of claim 1, wherein thehomogeneous solution comprises a mixture of gelatin and propylene glycolalginate.
 68. The process of claim 1, wherein the homogeneous solutioncomprises a mixture of atelocollagen, chondroitin sulfate and propyleneglycol alginate.
 69. A process of claim 1, wherein the homogeneoussolution comprises whey protein and propylene glycol alginate.
 70. Aprocess according to claim 1, wherein the homogeneous solution comprisesa mixture of ovalbumin and pectin.
 71. A process according to claim 1,wherein the homogeneous solution comprises a mixture of carboxymethylcellulose and propylene glycol alginate.
 72. A process according toclaim 1, wherein the substance comprises human serum albumin, gelatin,ovalbumin or carboxymethyl cellulose.